PROJECT SUMMARY Under stress conditions, the cells needs to rapidly respond to environmental clues to adapt the gene expression landscape. One particular aspect of the gene expression cascade that is emerging as a prime source for this rapid change is the manipulation of RNA turnover. Yet, what dictates RNA decay or stability in a time-sensitive and fast manner is not fully understood. In the recent years, post-transcriptional modifications have emerged as potent and dynamic regulator of a range of RNA functions including RNA stability. However, little is known about the regulation of post-transcriptional modifications in stress conditions or in response to rapid changes in gene expression. This proposal focuses on the post-transcriptional RNA modification N6- methyladenosine (m6A) and aims to determine how m6A status may control RNA fate in the face of widespread RNA decay. Our central hypothesis is that this modification helps discriminate mRNAs that are targeted for fast degradation from those that are spared. To test this hypothesis, we are using a powerful tool as we are taking advantage of a very potent viral nuclease. This endonuclease comes from the KSHV virus (Kaposi's Sarcoma Associated Herpesvirus) and has the ability, by itself, to trigger up to 80% of total mRNA degradation in mammalian cells. However, to date, it is unclear what renders an mRNA susceptible or resistant to this pervasive nuclease. In this proposal, we will use this viral system to query the host transcriptome response to this massive RNA decay event. We will combine RNA-seq and m6A-RIP seq strategies to assess how diverse the m6A landscape is among degraded or spared mRNAs. We will then monitor how the m6A machinery responds and/or is affected this re-structuring of the RNA steady state in the cell. In particular, our emphasis will be on the m6A readers that may directly be involved in decoding the m6A marks on the stable mRNAs. Finally, because RNA decay under stress conditions and/or in response to external stimuli is an heterogenous process, we will expand our exploration of m6A regulation of RNA stability to other sources of RNA degradation beyond viral nucleases using a novel site directed CRISPR Cas system. Taken together, we anticipate that these studies will shed light on a novel type of sensing mechanism that adapts the host cell environment to large changes in RNA stability. Since the regulation of RNA turnover is at the core many processes in the cell, understanding how post-transcriptional modifications may contribute to this complex balance should reveal novel pathways both in pathogenic and normal cells.